Minimal and hybrid hydrogenases are active from archaea.

Microbial hydrogen (H2) cycling underpins the diversity and functionality of diverse anoxic ecosystems. Among the three evolutionarily distinct hydrogenase superfamilies responsible, [FeFe] hydrogenases were thought to be restricted to bacteria and eukaryotes. Here, we show that anaerobic archaea encode diverse, active, and ancient lineages of [FeFe] hydrogenases through combining analysis of existing and new genomes with extensive biochemical experiments. [FeFe] hydrogenases are encoded by genomes of nine archaeal phyla and expressed by H2-producing Asgard archaeon cultures. We report an ultraminimal hydrogenase in DPANN archaea that binds the catalytic H-cluster and produces H2. Moreover, we identify and characterize remarkable hybrid complexes formed through the fusion of [FeFe] and [NiFe] hydrogenases in ten other archaeal orders. Phylogenetic analysis and structural modeling suggest a deep evolutionary history of hybrid hydrogenases. These findings reveal new metabolic adaptations of archaea, streamlined H2 catalysts for biotechnological development, and a surprisingly intertwined evolutionary history between the two major H2-metabolizing enzymes.

The maturase HydF enables [FeFe] hydrogenase assembly via transient, cofactor-dependent interactions.

[FeFe] hydrogenases have attracted extensive attention in the field of renewable energy research because of their remarkable efficiency for H2 gas production. H2 formation is catalyzed by a biologically unique hexanuclear iron cofactor denoted the H-cluster. The assembly of this cofactor requires a dedicated maturation machinery including HydF, a multidomain [4Fe4S] cluster protein with GTPase activity. HydF is responsible for harboring and delivering a precatalyst to the apo-hydrogenase, but the details of this process are not well understood. Here, we utilize gas-phase electrophoretic macromolecule analysis to show that a HydF dimer forms a transient interaction complex with the hydrogenase and that the formation of this complex depends on the cofactor content on HydF. Moreover, Fourier transform infrared, electron paramagnetic resonance, and UV-visible spectroscopy studies of mutants of HydF show that the isolated iron-sulfur cluster domain retains the capacity for binding the precatalyst in a reversible fashion and is capable of activating apo-hydrogenase in in vitro assays. These results demonstrate the central role of the iron-sulfur cluster domain of HydF in the final stages of H-cluster assembly, i.e. in binding and delivering the precatalyst.

Reversible or Irreversible Catalysis of H+/H2 Conversion by FeFe Hydrogenases.

Studies of molecular catalysts traditionally aim at understanding how a certain mechanism allows the reaction to be fast. A distinct question, which has only recently received attention in the case of bidirectional molecular catalysts, is how much thermodynamic driving force is required to achieve fast catalysis in either direction of the reaction. "Reversible" catalysts are bidirectional catalysts that work either way in response to even a small departure from equilibrium and thus do not waste input free energy as heat; conversely, "irreversible" catalysts require a large driving force to proceed at an appreciable rate [Fourmond et al. Nat. Rev. Chem. 2021, 5, 348-360]. Numerous mechanistic rationales for these contrasting behaviors have been proposed. To understand the determinants of catalytic (ir)reversibility, we examined the steady-state, direct electron transfer voltammetry of a particular FeFe hydrogenase, from Thermoanaerobacter mathranii, which is very unusual in that it irreversibly catalyzes H2 oxidation and production: a large overpotential is required for the reaction to proceed in either direction [Land et al. Chem. Sci. 2020, 11, 12789-12801]. In contrast to previous hypotheses, we demonstrate that in this particular enzyme catalytic irreversibility can be explained without invoking slow interfacial electron transfer or variations in the mechanism: the observed kinetics is fully consistent with the same catalytic pathway being used in both directions of the reaction.

B-factor Guided Proline Substitutions in Chromobacterium violaceum Amine Transaminase: Evaluation of the Proline Rule as a Method for Enzyme Stabilization.

Biocatalysis is attracting interest in the chemical industry as a sustainable alternative in large-scale chemical transformations. However, low operational stability of naturally evolved enzymes is a challenge and major efforts are required to engineer protein stability, usually by directed evolution. The development of methods for protein stabilization based on rational design is of great interest, as it would minimize the efforts needed to generate stable enzymes. Here we present a rational design strategy based on proline substitutions in flexible areas of the protein identified by analyzing B-factors. Several proline substitutions in the amine transaminase from Chromobacterium violaceum were shown to have a positive impact on stability with increased half-life at 60 °C by a factor of 2.7 (variant K69P/D218P/K304P/R432P) as well as increased melting temperature by 8.3 °C (variant K167P). Finally, the presented method utilizing B-factor analysis in combination with the proline rule was deemed successful at increasing the stability of this enzyme.

Photobiocatalytic CO2 reduction into CO by organic nanorod-carbon monoxide dehydrogenase assemblies: surfactant matters.

Photobiocatalytic CO2 reduction represents an attractive approach for conversion of solar light and abundant resources to value-added chemicals. However, the design of suitable systems requires a detailed understanding of the interaction between the artificial photosensitizer and biocatalyst interface. In this work, we investigate the effect of surfactant charge utilized in the preparation of a phenoxazine-based organic molecule nanorod photosensitizer on the interaction with the carbon monoxide dehydrogenase II from Carboxydothermus hydrogenoformans within biohybrid assemblies for sacrificially driven photobiocatalytic CO2 reduction into CO. Electrophoretic mobility shift assay in conjunction with cryogenic electron microscopy (Cryo-EM) and detailed physicochemical characterization are conducted to understand the interaction at the biohybrid interface in order to suggest a strategy for future functionalization of nanoparticles that fulfills the needs of the biocatalyst for green fuel production.

Secondary structure changes as the potential H2 sensing mechanism of group D [FeFe]-hydrogenases.

[FeFe]-hydrogenases function as both H2 catalysts and sensors. While catalysis is well investigated, details regarding the H2 sensing mechanism are limited. Here, we relate protein structure changes to H2 sensing, similar to light-driven bio-sensors. Our results highlight how identical cofactors incorporated in alternative protein scaffolds serve different functions in nature.

Probing Substrate Transport Effects on Enzymatic Hydrogen Catalysis: An Alternative Proton Transfer Pathway in Putatively Sensory [FeFe] Hydrogenase.

[FeFe] hydrogenases, metalloenzymes catalyzing proton/dihydrogen interconversion, have attracted intense attention due to their remarkable catalytic properties and (bio-)technological potential for a future hydrogen economy. In order to unravel the factors enabling their efficient catalysis, both their unique organometallic cofactors and protein structural features, i.e., "outer-coordination sphere" effects have been intensively studied. These structurally diverse enzymes are divided into distinct phylogenetic groups, denoted as Group A-D. Prototypical Group A hydrogenases display high turnover rates (104-105 s-1). Conversely, the sole characterized Group D representative, Thermoanaerobacter mathranii HydS (TamHydS), shows relatively low catalytic activity (specific activity 10-1 µmol H2 mg-1 min-1) and has been proposed to serve a H2-sensory function. The various groups of [FeFe] hydrogenase share the same catalytic cofactor, the H-cluster, and the structural factors causing the diverging reactivities of Group A and D remain to be elucidated. In the case of the highly active Group A enzymes, a well-defined proton transfer pathway (PTP) has been identified, which shuttles H+ between the enzyme surface and the active site. In Group D hydrogenases, this conserved pathway is absent. Here, we report on the identification of highly conserved amino acid residues in Group D hydrogenases that constitute a possible alternative PTP. We varied two proposed key amino acid residues of this pathway (E252 and E289, TamHydS numbering) via site-directed mutagenesis and analyzed the resulting variants via biochemical and spectroscopic methods. All variants displayed significantly decreased H2-evolution and -oxidation activities. Additionally, the variants showed two redox states that were not characterized previously. These findings provide initial evidence that these amino acid residues are central to the putative PTP of Group D [FeFe] hydrogenase. Since the identified residues are highly conserved in Group D exclusively, our results support the notion that the PTP is not universal for different phylogenetic groups in [FeFe] hydrogenases.

Chromobacterium violaceum ω-transaminase variant Trp60Cys shows increased specificity for (S)-1-phenylethylamine and 4'-substituted acetophenones, and follows Swain-Lupton parameterisation.

For biocatalytic production of pharmaceutically important chiral amines the ω-transaminase enzymes have proven useful. Engineering of these enzymes has to some extent been accomplished by rational design, but mostly by directed evolution. By use of a homology model a key point mutation in Chromobacterium violaceum ω-transaminase was found upon comparison with engineered variants from homologous enzymes. The variant Trp60Cys gave increased specificity for (S)-1-phenylethylamine (29-fold) and 4'-substituted acetophenones (∼5-fold). To further study the effect of the mutation the reaction rates were Swain-Lupton parameterised. On comparison with the wild type, reactions of the variant showed increased resonance dependence; this observation together with changed pH optimum and cofactor dependence suggests an altered reaction mechanism.

Light-Driven [FeFe] Hydrogenase Based H2 Production in E. coli: A Model Reaction for Exploring E. coli Based Semiartificial Photosynthetic Systems.

Biohybrid technologies like semiartificial photosynthesis are attracting increased attention, as they enable the combination of highly efficient synthetic light-harvesters with the self-healing and outstanding performance of biocatalysis. However, such systems are intrinsically complex, with multiple interacting components. Herein, we explore a whole-cell photocatalytic system for hydrogen (H2) gas production as a model system for semiartificial photosynthesis. The employed whole-cell photocatalytic system is based on Escherichia coli cells heterologously expressing a highly efficient, but oxygen-sensitive, [FeFe] hydrogenase. The system is driven by the organic photosensitizer eosin Y under broad-spectrum white light illumination. The direct involvement of the [FeFe] hydrogenase in the catalytic reaction is verified spectroscopically. We also observe that E. coli provides protection against O2 damage, underscoring the suitability of this host organism for oxygen-sensitive enzymes in the development of (photo) catalytic biohybrid systems. Moreover, the study shows how factorial experimental design combined with analysis of variance (ANOVA) can be employed to identify relevant variables, as well as their interconnectivity, on both overall catalytic performance and O2 tolerance.

Characterization of a putative sensory [FeFe]-hydrogenase provides new insight into the role of the active site architecture.

[FeFe]-hydrogenases are known for their high rates of hydrogen turnover, and are intensively studied in the context of biotechnological applications. Evolution has generated a plethora of different subclasses with widely different characteristics. The M2e subclass is phylogenetically distinct from previously characterized members of this enzyme family and its biological role is unknown. It features significant differences in domain- and active site architecture, and is most closely related to the putative sensory [FeFe]-hydrogenases. Here we report the first comprehensive biochemical and spectroscopical characterization of an M2e enzyme, derived from Thermoanaerobacter mathranii. As compared to other [FeFe]-hydrogenases characterized to-date, this enzyme displays an increased H2 affinity, higher activation enthalpies for H+/H2 interconversion, and unusual reactivity towards known hydrogenase inhibitors. These properties are related to differences in active site architecture between the M2e [FeFe]-hydrogenase and "prototypical" [FeFe]-hydrogenases. Thus, this study provides new insight into the role of this subclass in hydrogen metabolism and the influence of the active site pocket on the chemistry of the H-cluster.